The primary objective of this project is to study disease mechanisms of newly identified genes for vesicoureteral reflux (VUR) using a combined in vivo and in vitro molecular genetics approach. VUR is one of the commonest congenital anomalies of the kidney and urinary tract (CAKUT) in children. It is characterized by reflux of urine from the bladder into the kidney and is commonly associated with abnormal renal tract development, including defects in ureteric bud (UB) outgrowth and ureterovesical junction formation, and abnormal ureter development and peristalsis. Despite the high incidence of VUR in children, the genetic/molecular basis and disease mechanisms of VUR remain largely unclear. ROBO2 is one of few VUR/CAKUT genes that cause VUR phenotype in both human and animal model with many identified disease- causing mutations. Our recent studies show that mutations in SLIT2 and SRGAP1, two components of ROBO2 signaling pathway, can also result in CAKUT phenotype. However, the pathogenesis of SLIT2-ROBO2 signaling in VUR and CAKUT patients remain unclear. To test disease mechanisms and establish causality for ROBO2 mutations in human VUR, we will perform functional studies to determine if ROBO2 mutations disrupt interactions with known CAKUT-causing genes/proteins and if ROBO2 mutation knock-in mice also develop CAKUT and VUR phenotype (Aim 1). Since a recent study shows that ROBO2 controls UB outgrowth by regulating the separation of early Wolffian duct and nephrogenic cord, we will also study SLIT2 and SRGAP1 knockout mice to determine if they also play a role in early Wolffian duct and nephrogenic cord separation and if loss of SLIT2-ROBO2 signaling causes renal mesenchymal cell adhesion and migration defects (Aim 2). ZEB2 is a transcription factor that plays an import role in maintaining mesenchymal cells and promotes epithelial-mesenchymal transition via a BMP/SMAD/ZEB signaling network during development. Because we have recently found that deletion of ZEB2 gene can result in VUR phenotype in patients and ureter specific knockout of Zeb2 in mice causes abnormal ureter development, hydroureter, hydronephrosis and renal failure, we will also examine the role of Zeb2 in early ureteral mesenchymal cell development and determine if loss of Zeb2 in mouse ureter disrupts ureteral smooth muscle function and peristaltic machinery leading to VUR (Aim 3). Collectively, this research proposal will help us to understand disease mechanisms of newly identified VUR genes and define their molecular functions during urinary tract development. Results from this project will provide new knowledge in the molecular pathogenesis of VUR and assist us to identify novel therapeutic strategies for human VUR.